The major source of high-output nitric oxide (NO) during inflammation is inducible NO synthase (iNOS). Although iNOS is a transcriptionally-regulated generator of high NO, we have discovered a novel mode of post- translational, G protein coupled receptor (GPCR)-mediated activation of iNOS via ERK-dependent phosphorylation in endothelial cells. Post-translational activation of iNOS results in a further 3- to 5-fold increase in NO concentration over its already high basal amount. In Project 2, we will address the mechanisms by which iNOS phosphorylation leads to high output NO production and how this influences the lung endothelial barrier. We will test the hypotheses that (i) activation of the kinin GPCR, BI, induces ?-arrestin2 scaffolding of iNOS and ERK, which in turn phosphorylates and activates iNOS due to enhanced dimerization and isomerization by the prolyl isomerase Pin1, (ii) S-nitrosylation of ?-arrestin2 dissociates it from iNOS, resulting in dephosphorylation and inactivation ,and (iii) post-translationally activated iNOS-derived NO causes increased lung vascular permeability in the context of NADPH oxidase (NOX2) activation and peroxynitrite generation resulting in endocytosis of VE-cadherin and inactivation of p190RhoGAP. The signaling pathways mediating receptor-dependent post-translational activation of iNOS in pulmonary endothelial cells and its effects on pulmonary vascular permeability will be investigated using imaging, cell biology, biochemical, and physiological approaches. We will thereby establish the role of key signaling molecules ?-arrestin2 and Pin1 in mediating INOS activation and functions of ?-arrestin2, p190RhoGAP and VE-cadherin in mediating disruption of lung endothelial barrier function. The proposed studies we hope will provide novel therapeutic strategies to inhibit detrimental consequences of high concentrations of NO in inflammatory lung injury based on the deeper understanding of signaling pathways by which iNOS is activated secondary to its expression in the endothelium.